Proteins and peptide derivatives can currently be displayed on the surface of bacteria, viruses, and yeast for a variety of biotechnological applications such as biopharmaceutical development, peptide discovery, and basic research. Phage display remains the most widely used method for expressing protein libraries. Bacteriophage (phage) display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides for ones with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. U.S. Pat. Nos. 5,223,409; 5,403,484; 5,571,689; 5,663,143.
More recently, eukaryotic and prokaryotic expression systems have been considered for peptide display. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts with E. coli being the primary expression host studied. Cell surface display is attractive since fluorescence activated cell sorting (FACS) can be applied for sensitive, quantitative library analysis and screening. Furthermore, cell display enables simple clonal and library manipulation and propagation and direct measurement of the relative binding constants of isolated clones. However, the general utility of bacterial display for screening peptide and protein libraries is currently limited by the suitability of typical display scaffolds—including outer membrane proteins, fimbria (FimH), and flagella (FLITRX). While such systems have had some success in antibody and protein epitope mapping experiments, less success has been achieved in the isolation of soluble affinity reagents, which typically requires grafting of the peptides into constrained, soluble scaffold proteins. Given these problems, bacterial display systems have not proven generally useful for generating affinity reagents.
In addition to the above problems with prior cell display systems, each suffers from usefulness in limited temperature ranges that are amenable to host cell viability (typically 20-40° C.). As such, new methods and compositions are required for peptide and protein display.